Intracellular manganese ions (Mn) and the enzyme Cu/Zn superoxide dismutase (SOD1) have overlapping roles in oxidative stress protection. While the mechanism of SOD1 action in superoxide detoxification has been well characterized, very little is understood about how cells utilize Mn to suppress oxidative damage independent of SOD enzymes. Recently, using S. cerevisiae as a model organism, we have reported that proper phosphate metabolism is important for suppressing oxidative damage and critical for enabling cells to utilize Mn as an antioxidant. It was found that sod1 null stains engineered to hyperaccumulate phosphate are oxidatively stressed and inviable in air. Preliminary results indicate that high cytoplasmic polyphosphate (PolyP) is responsible for the severity of oxidative damage and phosphate interactions with both Mn and Fe are involved. We hypothesize that PolyP enhances oxidative injury by sequestering Mn and Fe, thereby limiting their availability to the Mn-antioxidant and to essential Fe/S proteins that are susceptible to oxidative injury. The purpose of the current proposal is to test this hypothesis and elucidate the nature of the Mn-antioxidant. In order to determine the role of PolyP in oxidative stress, a series of yeast strains that have altered PolyP metabolism will be engineered. These strains, hereafter referred to as the polyphosphate titratable series (PTS), which will have variations in the size, content, and cellular localization of PolyP, will be exploited to assess the impact of PolyP on various indicators of oxidative stress and on Mn and Fe bioavailability. In the sod1 null background, the PTS strains can be used to determine how PolyP influences Mn-suppression of oxidative damage and Fe availability for repairing damaged Fe/S clusters. Furthermore, we will directly monitor Mn- and Fe-PolyP interactions inside the PTS mutants as a function of oxidative stress resistance by using a newly developed application of ENDOR spectroscopy to whole cells. In toto, these experiments will reveal exactly how polyphosphate influences oxidative stress and the role Mn and Fe play in mediating its toxicity. In addition, the mechanism of Mn suppression of oxidative stress will be determined by employing a high-throughput genetic screen to identify genes that are required for Mn-antioxidant activity. sod1 null yeast will be mutagenized with a transposon library and mutants that exhibit loss of Mn rescue of oxidative damage will be selected. This screen is designed to select for genes that are involved in the metabolism of small molecules that bind and activate Mn for Mn-antioxidant activity. Overall, these studies should provide great insight into the role of phosphate, Mn, and Fe in cellular oxidative stress and the factors that govern Mn suppression of oxidative damage. Studies of this type are at the heart of understanding and perhaps treating the numerous human disorders attributed to oxidative stress. PUBLIC HEALTH RELEVANCE: Damage from oxygen radicals has been linked to a number of human diseases, including reperfusion injury, cancer, cardiovascular disease, neurological degeneration, and aging. Studies into the basic mechanisms of cellular oxidative stress resistance are crucial towards understanding the role of oxygen radicals in disease and to the eventual development of therapeutic strategies. We have recently shown that, in addition to superoxide dismutase (SOD) enzymes, manganese is critical for sustaining life in atmospheric oxygen. However, very little is understood about how cells utilize Mn as an antioxidant. The purpose of the current investigation is to decipher the mechanism of cellular Mn-antioxidant activity, with a particular emphasis on the interplay between phosphate, Mn and Fe metabolism.